This project focuses on the biochemical characterization of a series of single chain ribosome inactivating proteins (SCRIPs) of plant origin, which also inhibit infection and replication of human immunodeficiency virus (HIV-1). During this reporting period, GAP 31 from the seeds of Gelonium multiflorum, and DAP 30 and DAP 32 from pink carnation leaves (Dianthus caryophyllus) have been purified to homogeneity. They are basic proteins, 31, 30, or kDa, respectively in size and exhibit dose- dependent inhibition of cell free HIV-1 infection ad replication as measured by syncytium formation, p24 expression and HIV reverse transcriptase activity in HIV-1 infected H9 T-lymphocytes. No toxicity to cellular DNA and protein synthesis of intact host cells can be detected at the dose level 3-4 orders of magnitude higher than the level effective against the infection and replication of HIV. The sequence analyses of the N-terminal 60 amino acid residues have revealed little homology to previously known anti-HIV/SCRIPs. However, the homology can be aligned at Phe, Tyr, Ser/Thr. A basic residue at the unique positions 10-16 is also present consistent with the notion that it has an important role in reducing cytotoxicity of these proteins toward host cells. A significant homology was found between residues 1-40 of GAP 31 and 660-699 of Drosophila DNA topoisomerase (TIase) II. This segment is conserved between TIase II and other TIases. Indeed, GAP 31, DAP 30 and DAP 32 irreversibly relax and decatenate supercoiled DNA and catalyze double-stranded breakage to form linear DNA. The relaxed products are topologically inactive and no longer serve as substrate for DNA gyrase, a phenomenon similar to that of cellular TIase in the presence of TIase poisons. The ability of these anti-HIV agents to interrupt essential topological interconversions of DNA suggests that their anti-HIV activity may not be merely a consequence of ribosome inactivation. Dual capability to act on DNA and RNA modifications may provide a novel mechanism for their antiviral action. A rat brain ADP-ribosylarginine hydrolase was sequenced for the N- terminal portion and fragments from BrCN and trypsin cleavages. Based on these amino acid sequences, a rat brain Lambda ZAP library was screened using oligonucleotide and PCR-generated cDNA probes which permitted the assembly of a 1086-base pair open reading frame. The deduced protein sequence contains amino acid sequences found in the purified 39 kDa hydrolase. By the same methodology, amino acid sequences of tryptic fragments from a guinea pig pregnenolone-binding protein were obtained for the cloning of a cDNA whose sequence deduced to a 34 kDa protein corresponding to adrenocortical estrogen sulfotransferase.